Ceftaroline Susceptibility among Isolates of MRSA: A Comparison of EUCAST and CLSI Breakpoints

Background Methicillin-resistant Staphylococcus aureus (MRSA) is an important bacterial pathogen causing a number of community-acquired and nosocomial infections. Ceftaroline fosamil is a fifth generation cephalosporin, approved for the treatment of infections caused by MRSA. The main objective of this study was to estimate the susceptibility of ceftaroline among isolates of MRSA by using CLSI and EUCAST breakpoints. Materials and Methods Fifty non-duplicate isolates of MRSA were included in the study. Ceftaroline susceptibility was done using E-strip test and interpreted using CLSI and EUCAST breakpoints. Results Susceptible isolates were equal (42%) by both CLSI and EUCAST, while resistant isolates were more commonly seen in EUCAST (50%). Ceftaroline MIC ranged from 0.25- >32µg/ml. All the isolates were sensitive to Teicoplanin and Linezolid. Conclusions Resistant isolates were less (30%) while using the CLSI 2021 criteria probably due to the inclusion of SDD category. Our study showed that Fourteen isolates (28%) had Ceftaroline MIC >32µg/ml, which is an alarming finding. The high percentage of Ceftaroline resistant isolates in our study probably suggest a hospital spread of Ceftaroline resistant MRSA emphasizing the need for stringent infection control precautions.


INTRODUCTION
Methicillin-resistant Staphylococcus aureus (MRSA) is an important bacterial pathogen causing a number of communityacquired and nosocomial infections, including septicemia, skin and soft tissue infections, osteomyelitis, and endocarditis (1). It is a global health problem and the prevalence of MRSA in India ranges from 25-50% (2). Misuse of Antibiotic has led to high resistance levels in MRSA strains resulting in an increased mortality rate, high costs of care and treatment, and longer periods of hospitalization (3). The emergence of highly virulent community-associated MRSA (CA-MRSA) causing skin infections, sepsis, toxic shock syndrome, and necrotizing pneumonia is a major concern (4). The drug of choice for treating severe MRSA infections is Vancomycin. therapeutic index, slow bactericidal activity, difficulty in achieving pharmacokinetic and pharmacodynamic targets and potential side effects like nephrotoxicity and ototoxicity (5,6). There are also reports of treatment failures with vancomycin in critically ill patients due to suboptimal therapeutic levels or high MIC(Minimum Inhibitory Concentration) values (7). Alternative drugs like Teicoplanin, linezolid and daptomycin are being increasingly used for treatment of MRSA infections (8).
MRSA is frequently isolated from complicated skin and soft tissue infections and the cases of MRSA are increasing among outpatients (9,10,11,12). Ceftaroline fosamil is a fifth generation cephalosporin active against methicillin-susceptible (MSSA) and MRSA. It was approved in October 2010 by the United States Food and Drug Administration (US FDA) for the treatment of adults with communityacquired bacterial pneumonia and acute bacterial /skin and skin structure infections (ABSSSI)(13). Ceftaroline acts by inhibiting cell wall synthesis by binding to Penicillin Binding Proteins (PBP) 1, 2, 3 and PBP 2a for MRSA (14). Studies have shown that the drug is well tolerated by patients and is as effective as vancomycin, daptomycin and linezolid in eradicating MRSA (15,16). Resistance to ceftaroline is uncommon but several studies have reported decreased susceptibility of MRSA to ceftaroline in sporadic cases. The resistance may be due to the mutation within PBP 2a protein, in particular, outside the Penicillin-Binding Domain (nPBD) (17,18).
Ceftaroline has been approved for the treatment of cSSTI (Complicated Skin and Soft tissue infections) at a standard dosage of 600 mg every 12 h (given over 60 min) in adults (19). In 2017, the European Medicines Agency approved a higher dosing regimen of ceftaroline (600 mg every 8 h over 120 min) for cSSTI caused by S. aureus with a ceftaroline MIC of 2 or 4 mg/L. European Committee on Antimicrobial Susceptibility Testing (EUCAST) introduced an intermediate breakpoint of 2 mg/L for ceftaroline against S. aureus, for indications other than pneumonia, in version 8.0 of the EUCAST breakpoints due to the approval of a higher dosing regimen by the European Medicines Agency. As a result, the EUCAST resistant breakpoint for ceftaroline against S. aureus increased from >1 mg/L in version 7.1 of the breakpoint tables to >2 mg/L in version 8.0(20,21,22).
Clinical Microbiology laboratories in several countries use breakpoints published by CLSI (Clinical Laboratory Standards Institute) for susceptibility testing. In January 2019, CLSI also modified the ceftaroline breakpoints and introduced the susceptible dose dependent (SDD) category for this agent, based on the recommendation by the European Medicines Agency in 2017 (High dose regimen of 600 mg every 8 h over 120 min), although this dosing regimen is not approved by the US Food and Drug Administration(23). There are very few studies in India evaluating the susceptibility of S.aureus to ceftaroline and there is very little data available about the susceptibility pattern of S.aureus to ceftaroline (24,25,26). The aim of this study was to: 1. Estimate the rate of in vitro susceptibility of ceftaroline among isolates of MRSA by Etest strip using CLSI and EUCAST breakpoints; 2. To assess the agreement between the two guidelines for susceptibility testing of Ceftaroline; and 3. To find the antimicrobial susceptibility pattern of MRSA isolated during the study period. Testing for ceftaroline susceptibility was done by E-test strip method. The ceftaroline Etest strips (0.002-32 µg/mL) was obtained from Biomerieux, France. The E-test strips were placed on the lawn culture of the organism and the plates were incubated at 37°C for 18-24 hours. MIC's were read where the ellipse intersects the MIC (Minimum Inhibitory Concentration) scale. Since E-test strip has continuous gradient, MIC values "in-between" two-fold dilutions can be obtained. These values were rounded up to next two-fold dilution before categorisation. MICs were interpreted according to EUCAST version 11.0 and CLSI 2021. EUCAST version 11.0 for Ceftaroline (In pneumonia) -≤1-Susceptible, > 1-Resistant EUCAST version 11.0 for Ceftaroline (For conditions other than pneumonia)-

RESULTS
Among the 50 isolates of MRSA, 26 isolates were from samples received from the Surgery department, while 18 and 6 were from Medicine and Obstetrics departments respectively. Of the 50 patients, 30 were admitted in the ward while ten patients were admitted in Intensive care. The remaining ten samples were from patients attending the Outpatient Department. Among the 50 isolates of MRSA, Ceftaroline susceptibility by CLSI and EUCAST (Table 1). Susceptible isolates were equal (42%) by both CLSI and EUCAST, while resistant isolates were more commonly seen in EUCAST (50%). Ceftaroline MIC ranged from 0.25->32µg/ml. An Isolate of MRSA with Ceftaroline MIC >32µg/ml ( Fig. 1). Twenty-one isolates were susceptible by both CLSI and EUCAST and had MIC ranging from 0.25-0.50 µg/ml (  Fourteen isolates (28%) had Ceftaroline MIC >32µg/ml. The MIC50 of the isolates was 2µg/ml. Antimicrobial susceptibility pattern of the isolates (Table 3). All the isolates were sensitive to Teicoplanin and Linezolid. Majority of the isolates were also sensitive to Gentamicin, Tetracycline and Cotrimoxazole.  35,36). MIC50 of the isolates in the studies conducted by Gaikwad and Basireddy et al were 0.38µg/ml and 0.5µg/ml respectively which was discordant with the findings in our study, which showed an MIC 50 of 2 µg/ml (24,26). The MIC distribution of the isolates in our study were discordant with the study conducted in Turkey which found that 94.3% of tested MRSA isolates were inhibited by ceftaroline (MIC≤1 µg/mL) (37).In our study all the 50 isolates(100%) were susceptible to Teicoplanin and Linezolid, while the resistant rates to Erythromycin, Clindamycin and Ciprofloxacin were 66%, 48% and 56% respectively. This finding was quite similar to the study conducted by Elfeky et al who showed that 63% of the isolates were resistant to Erythromycin, Clindamycin and Ciprofloxacin (38). Susceptiblity pattern of the 14 isolates with Ceftaroline MIC >32µg/ml were not completely identical. Among these 14 isolates, 85.7% were sensitive to Gentamicin and Tetracycline, while 21.4% were sensitive to Erythromycin and Ciprofloxacin. In addition, 71.4% of these isolates were sensitive to Cotrimoxazole, while 35.7% of these isolates were susceptible to Clindamycin. Among the 14 isolates, 50% were from patients admitted in surgical department, while 42.9% were from patients in general medicine department. In conclusion, in this study Ceftaroline susceptibility in MRSA was evaluated using CLSI and EUCAST. Only 42% of the isolates were Ceftaroline susceptible by both guidelines. Resistant isolates were less (30%) while using the CLSI 2021 criteria probably due to the inclusion of SDD category. Ceftaroline MIC ranged from 0.25->32µg/ml .Our study showed that Fourteen isolates(28%) had Ceftaroline MIC >32µg/ml,which is an alarming finding. The high percentage of Ceftaroline resistant isolates in our study probably suggest a hospital spread of Ceftaroline resistant MRSA emphasizing the need for stringent infection control precautions. To the best of our knowledge this is the first study from India showing a high percentage of Ceftaroline resistance,probably suggesting that Ceftaroline is not a good treatment option for MRSA infections in our hospital.
The study had a relatively small sample size .Genome sequencing of the isolates could not be done to look for any mutations in PBP2a. Epidemiologic characterization of the isolates could not be done to look for any clonal transmission.